The use of microorganisms to conduct modification of biomass for the production of bioenergy products or metabolites has been proposed in the art. Such process, ideally, would require two major types of activities: (i) a degradation activity, to transform biomass into fermentable sugars and (ii) a fermentation activity, to transform said sugars into bioenergy products or other valuable metabolites. So far, efforts have been directed mainly towards the identification of microorganisms having the ability to catalyze the fermentation step.
A monograph on the production of ethanol through fermentation with microorganisms was published under the title “Ethanol Fermentation Strains” by J. R. Hettenhaus, under the aegis of the United States Department of Energy and the National Renewable Energy Laboratory (Dec. 16, 1998). In this document, which summarizes the contributions made by participants in the study, it is pointed out that:                only microorganisms similar to Saccharomyces, Zymomonas and E. coli can be used in existing equipment;        in the short term, the increased fermentation of xylose and arabinose could be the main objective, it being specified however that it is of little interest to increase the converting efficacy of the other sugars of hexose or oligomer type;        over the longer term, gains could be achieved regarding higher operating temperatures and combining of the steps of enzyme production, saccharification and hydrolysis.        
Current industrial processes only allow the culture and growth of microorganisms for the fermentation and extraction of ethanol at temperatures in the region of 30° C., owing to the fragility of the industrial microorganisms (yeasts) used. They also entail major bioenergy costs to concentrate the ethanol after fermentation, since yeasts currently used for this fermentation cannot withstand ethanol concentrations above 100 g/l. Additionally, the fermentation of yeasts practically only uses C6 sugars, of glucose type.
The conversion of biomass using microorganisms has also been tested (Blumer-Schuette et al., 2008, Extremely thermophilic microorganisms for biomass conversion: status and prospects, Curr Opinion Biotechnol 19, pp. 210-217; Perez et al., 2002, Int Microbiol 5, pp 53-63). However, as reported in Mosier et al. (Bioresource Technology 96 (2005) 673-686), a pre-treatment of lignocellulosic biomass is required to alter the structure of cellulosic biomass to make cellulose more accessible to the enzymes that convert the carbohydrate polymers into fermentable sugars.
The industrial and efficient production of fermentable sugars (e.g., monomeric sugars) from raw (i.e., starch, lignocellulosic) biomass still remains a challenge. Various approaches have been proposed to exploit raw biomass, such as thermochemical methods, acid hydrolysis or enzymatic hydrolysis. Sun H et al (Appl Biochem Biotechnol. February 2010; 160(4):988-1003) discusses the use of non Deinococcus enzymes for the degradation of starch. Polizeli M L et al, and Collin T et al (Appl Microbiol Biotechnol. June 2005; 67(5):577-591; FEMS Microbiol Rev. January 2005; 29(1):3-23) reviews the use of non Deinococcus enzymes for the degradation of xylan. Wilson D B et al (Curr Opin Biotechnol. June 2009; 20(3):295-299) discusses the use of non Deinococcus enzymes for the degradation of cellulose for biofuel applications.
These approaches, however, did not lead so far to the implementation of an effective and industrial enzymatic method of producing metabolites from biomass. Furthermore, due to the wide diversity of lignocellulosic biomass, with each having a specific composition of starch, cellulose, hemicellulose and lignin, there is a need for additional enzymes with improved activities.
Accordingly, there is a need for novel enzymes active in the modification of biomass. There is also a need in the art for a cost-effective and scalable process for the degradation of starch and lignocellulosic biomass into valuable products such as fermentable sugars or bioenergy products and metabolites.
Work conducted by the applicant has led to the surprising finding that strains of the genus Deinococcus exhibit remarkable properties for use in the transformation of biomass into metabolites, including bioenergy products (WO2009/063079). More particularly, the applicant has demonstrated that Deinococcus strains are able to catalyse or cause biomass degradation into fermentable sugars, and to produce metabolites from said sugars. Applicant has also demonstrated these strains are resistant to and active at elevated temperature, elevated ethanol concentrations, and within a wide range of pH values (PCT/EP2010/056600, presently unpublished). The applicants have further discovered that Deinococcus bacterium may degrade raw biomass, including starch, xylan or cellulose, which provides additional substantial advantages for biomass conversion and metabolite production (WO2010/094665). Deinococcus, as well as related bacteria, therefore open the path towards new and efficient bioenergy and metabolite production from biomass.
The present invention discloses novel enzymatic activities derived from Deinococcus and related bacteria. These enzymes are involved in energetic metabolism. They have been structurally characterized and exhibit distinct motifs or sequences, which confer on said proteins remarkable biological activities. These Deinococcal enzymes have the ability e.g., to hydrolyse the main constituents of biomass, including xylan, cellulose, and/or hemicelluloses or any lignocellulosic material under conditions suitable for an industrial process. Such enzymes had never been reported or isolated in the art and bring substantial improvements to the development of industrial processes of transforming biomass. The enzymes may be used as such, in purified or isolated form, or in a mixture of enzymes comprising at least one enzyme of this invention, in industrial processes. These enzymes also allow or favour the use of cheaper carbon source for fermentation and may be used in the production of metabolites, bioenergy products, or in the production (or over-expression) of recombinant proteins.